ABSTRACT
Objective@#To evaluate the differential calretinin immunostaining in different segments of total colonic aganglionosis and its utility in the diagnosis.@*Methods@#Nine specimens including ileum and colon segments were obtained from 9 patients with total colonic aganglionosis (TCA), from 2010 to 2016 year, in Wuhan Children′s Hospital, Tongji Medical College, Huazhong University of Science and Technology. Another 9 ganglionic specimens including the same segments from patients with non-Hirschsprung disease (non-HD) patients were collected as control. All cases were immunostained with calretinin. The patterns of calretinin immunostaining were observed, and morphometric analysis of each sample was performed by image analysis program (Image-Pro-Plus). The mean absorbance was evaluated by calculating the areas of the lamina propria occupied by the positively stained area of the calretinin at high power field.@*Results@#The same pattern of calretinin immunostaining was seen in ganglionic ileum and ganglionic colon segments, with staining seen in intrinsic nerves fibers (INF), and in granular aggregates in the lamina propria and muscularis mucosae. There was no significant difference in the numbers of calretinin-positive INF from the ganglionic segments. In contrast, the number of calretinin-positive INF and granular aggregates in aganglionic segments were significantly lower than those in the ganglionic group (P<0.01). In the ileum transitional zone, scattered calretinin staining was observed, and the amount of calretinin-positive INF was significantly lower than those in the proximal segment of ganlionic ileum (P<0.01).@*Conclusions@#Since there is significant different expression of calretinin among the different segments from TCA, calretinin immunostaining has potential value in detecting TCA. It could be an important adjunctive method in detecting TCA in the future.
ABSTRACT
BACKGROUND: How to establish a stable in vitro culture system, including location of corneal limbal epithelial stem cells, in vitro sample harvest, in vitro culture, vector selection, as well as identification methods, play a key role in corneal limbal epithelial stem cells culture. OBJECTIVE: To culture the isolated rabbit corneal limbal epithelial stem cells and to identify the biological properties of cultured cells. METHODS: The primary rabbit cornel limbal epithelial stem cells were isolated and cultured with tissue inoculation using human amniotic membrane as vector. The growth features of cells were observed under an inverted microscope. The morphology of cells was observed by hematoxylin-eosin staining and a scanning electron microscope. Furthermore, the monoclonal antibody AE5 and P63 two-step immunohistochemical staining were used to identify limbal epithelial stem cell protein expression. RESULTS AND CONCLUSION: The rabbit corneal limbal epithelial stem cells could be successfully cultured and maintained a relatively high value-added potential in vitro. Rabbit corneal limbal epithelial stem cells cultured on the amniotic membrane pull netted cellular layer. The AE5 monoclonal antibody positive rate of primary cultured cells was about 5% and P63 monoclonal antibody positive up to 90%. AE5-positive rate increased and P63-positive rate decreased with the increase in the number of subculture. The rabbit limbal epithelial stem cells can be successful culture and amplified on human amniotic membrane in vitro by limbal tissue culture method. The cultured cells maintain the characteristics of corneal epithelial cells. The rabbit corneal limbal epithelial stem cells can form grafts on the amniotic membrane.